Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans

Abstract

Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol ⌬ 5,6 -desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, >256, 16, 16, 8, and 1 g ml ؊1 , respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 g ml ؊1 ); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 g ml ؊1 ). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14␣-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target (Erg11p) Modifications

“…However, cells can overcome the effect of such a block by a suppressor mutation in sterol C5,6 desaturation and can thus acquire azole resistance (69). In agreement with this observation, azole-resistant fungal isolates with nonfunctional ERG3 alleles have been described for C. albicans and Candida dubliniensis (70)(71)(72). Both strains with a putative block at the Erg11p level (CL-6835 and ATCC 200956) carried ERG3 mutations (Table 1) and showed an accumulation of 24-methylcholesta-5,7,24(28)-trien-3-ol ( Table 2), suggesting that these two strains might have evolved to avoid the production of toxic sterols like 14,24-dimethylcholesta-8,24(28)-3␤,6␣-diol (Fig.…”

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

“…Heeres et al (63) also determined the MIC 100 for clotrimazole with C. albicans to be Ͼ100 g ml Ϫ1 in Sabouraud broth, which was surprisingly high considering that clotrimazole is used for the treatment of Candida infections in humans and calls the methodology into question. More recent MIC determinations for clotrimazole against C. albicans range from 0.03 to 1 g ml Ϫ1 (64)(65)(66), suggesting that the MIC determinations reported by Heeres et al (63) were over 100-fold higher than present determinations. In contrast, Marking et al (67) found the MIC for clotrimazole to be 10 to 30 g ml Ϫ1 after a 15-min exposure, which fell to 10 g ml Ϫ1 after 60 min with Saprolegnia hypogyna.…”

Genetic and phenotypic characterization of Candida albicans strains isolated from infectious disease patients in Shanghai

“…In addition, their ERG11 sequences did not reveal mutations unique to resistant strains. It is possible that other genes, such as ERG5 and ERG3, contribute to azole resistance (Martel et al, 2010a(Martel et al, , 2010b, which deserves further investigation.…”